Method for sorting antifungal molecules acting on the glucanosyltransferase activity

ABSTRACT

The invention discloses proteins having β (1–3)glucanosyltransferase type activities. These proteins can be used for detecting the antifungal activity of molecules.

This is a division of application Ser. No. 09/242,913, filed Oct. 13,1999, which issued as U.S. Pat. No. 6,551,811 on Apr. 22, 2003, which isa Section 371 application of PCT/FR97/01 540, filed Aug. 29, 1997, andfurther claims the benefit of U.S. Provisional Application No.60/024,910, filed Aug. 30, 1996, the disclosures of all of which areincorporated herein by reference.

This invention relates to a protein with an activity of theglucanosyltransferase type, and more especially aβ-(1–3)-glucanosyltransferase activity.

This invention also relates to oligonucleotides coding for this proteinhaving an enzymatic activity.

It also relates to molecules having an effect on the activity of thisenzyme.

Opportunistic fungal infections due to Candida, Aspergillus,Cilpuococcus and Pneumocystis are responsible for the increase inmorbidity and mortality among patients suffering from AIDS and otherpatients with clinically compromised immunity. In addition, the yeastCanidida and the dermatophytes today remain a major medical problemamongst patients with adequate immunity. Despite the increase in thenumber of infections due to pathogenic and opportunistic fungi, therapyagainst mycoses has not improved in recent years. Two families of drugsare used: the azoles and Amphotericin B. These drugs have somedisadvantages since treatment based on Amphotericin B is associated withnephrotoxicity and that based on azole is more fungistatic thanfungicidal.

Fungi are microorganisms of the eukaryotic type which share the majorityof their biochemical pathways with their hosts, with one importantexception: the biosynthesis of the cell wall. The cell wall is a rigidenvelope which protects the cell against the environment and mechanicalstresses, but is also a dynamic structure which is involved in thetransport of ions and macromolecules and in the localization of enzymesinvolved in fungal growth. In consequence, disorganization of theorganization of the cell wall should be detrimental to fungi.

The skeleton of the fungal cell wall is mainly composed of polymers ofthe polysaccharide type (β(1–3) glucans, mannans, chitin) which are notfound in humans. For this reason, the biosynthesis of the cell wall hasbeen a target for research into new antifungal drugs. The penicillinsand cephalosporins, which are both inhibitors of the bacterial cellwall, and potential antibiotics lend support to this hypothesis.Moreover, many molecules which inhibit the development of the fingalcell wall have antifungal properties (Debono and Gordee, 1994, Annu.Rev. Microbiol, 48, 471–497). Among these are:

-   -   1) The families of the echinocandin lipopeptides and the        palulacandin glycopeptides which are non-competitive inhibitors        of the glucan synthetase complex.    -   2) The polyoxins and nikkomycins which are analogs of UDP-GlcNac        and potential competitive inhibitors of chitin synthetase, and    -   3) The pradimycins binding mannan and the benanomycins.

The synthesis of β (1–3) glucan and chitin is under the control ofenzyme complexes (glucan synthetase and chitin synthetase) which arelocalized in the plasma membrane. Once the polymers have been releasedinto the periplasmic space, cross-links are created between the polymersand it is these which are responsible for the rigidity of the cell wall.The proteins and genes of the glucan and chitin synthetases arebeginning to be fairly well understood.

However, the inhibition of the glucan and chitin synthetases by amolecule requires three steps : its transfer across the cell wall,crossing of the plasma membrane and transfer inside the cell to thetarget, each step representing a potential barrier for the enzymaticinhibitor from being an effective antifungal drug, or a potential sourceof resistant strains against the drug.

The transferases which are responsible for creating the covalent bondsbetween the different polymers of the wall have been very little studiedup till now.

These enzymes represent a better target than the chitin and glucansynthetase complexes since they are more easily accessible for aputative antifungal drug.

Nuoffer et al. (1991, Mol. Cell. Bio., 11, 27–37) have described aglycoprotein, named Gaslp, exposed on the surface of the yeastSaccharomyces cerevisiae. The genes coding for this protein have beencloned. The function of the Gaslp protein is not. essential for theviability of the cell, and has not been determined.

Saporito-Lrig et al. (1995, Mol. Cell Biol., 15, 601–613) have isolateda gene originating from the yeast Candida albicans, designated PHR1. Theamino acid sequence determined for this protein PIR1 was 56% identicalto that of the protein Gasl. The gene was regulated in response to thepH of the culture medium. As for the protein gaslp, no function has beendetermined.

It clearly emerges from this analysis of the prior art that there hasbeen a problem in obtaining molecules with effective antifingalactivity.

The inventors have solved this problem.

They have shown that the introduction of mutations into aglucanosyltransferase originating from Aspergillus fumigatus interfereswith the development of this micro-organism.

They have also determined the sequences of several of these enzymes.

The present invention thus relates to a first protein with an activityof the β-(1–3)-glucanosyltransferase type characterized in that it hasat least 50%, preferably 60%, and even more preferably 85% homology withproteins having the sequences, or a part of the sequences SEQ ID NO 2 orSEQ ID NO 3 as follows:

Met Lys Ala Ser Ala Val Thr Ala Ala Leu Ala Val Gly Ala Ser Thr SEQ IDNO:2 Val Leu Ala Ala Pro Ser Ile Lys Ala Arg Asp Asp Val Thr Pro Ile ThrVal Lys Gly Asn Ala Phe Phe Lys Gly Asp Glu Arg Phe Tyr Ile Arg Gly ValAsp Tyr Gln Pro Gly Gly Ser Ser Asp Leu Ala Asp Pro Ile Ala Asp Ala AspGly Cys Lys Arg Asp Ile Ala Lys Phe Lys Glu Leu Gly Leu Asn Thr Ile ArgVal Tyr Ser Val Asp Asn Ser Lys Asn His Asp Glu Cys Met Asn Thr Leu AlaAsp Ala Gly Ile Tyr Leu Val Leu Asp Val Asn Thr Pro Lys Tyr Ser Ile AsnArg Ala Lys Pro Lys Glu Ser Tyr Asn Asp Val Tyr Leu Gln Tyr Ile Phe AlaThr Val Asp Ala Phe Ala Gly Tyr Lys Asn Thr Leu Ala Phe Phe Ser Gly AsnGlu Val Ile Asn Asp Gly Pro Ser Ser Ser Ala Ala Pro Tyr Val Lys Ala ValThr Arg Asp Leu Arg Gln Tyr Ile Arg Ser Arg Lys Tyr Arg Glu Ile Pro ValGly Tyr Ser Ala Ala Asp Ile Asp Thr Asn Arg Leu Gln Met Ala Gln Tyr MetAsn Cys Gly Ser Asp Asp Glu Arg Ser Asp Phe Phe Ala Phe Asn Asp Tyr SerTrp Cys Asp Pro Ser Ser Phe Lys Thr Ser Gly Trp Asp Gln Lys Val Lys AsnPhe Thr Gly Tyr Gly Leu Pro Leu Phe Leu Ser Glu Tyr Gly Cys Asn Thr AsnLys Arg Gln Phe Gln Glu Val Ser Ser Leu Tyr Ser Thr Asp Met Thr Gly ValTyr Ser Gly Gly Leu Val Tyr Glu Tyr Ser Gln Glu Ala Ser Asn Tyr Gly LeuVal Glu Ile Ser Gly Asn Asn Val Lys Glu Leu Pro Asp Phe Asp Ala Leu LysThr Ala Phe Glu Lys Thr Ser Asn Pro Ser Gly Asp Gly Asn Tyr Asn Lys ThrGly Gly Ala Asn Pro Cys Pro Ala Lys Asp Ala Pro Asn Trp Asp Val Asp AsnAsp Ala Leu Pro Ala Ile Pro Glu Pro Ala Lys Lys Tyr Met Thr Glu Gly AlaGly Lys Gly Pro Gly Phe Ala Gly Pro Gly Ser Gln Asp Arg Gly Thr Gln SerThr Ala Thr Ala Glu Pro Gly Ser Gly Ser Ala Thr Gly Ser Ser Ser Ser GlyThr Ser Thr Ser Ser Lys Gly Ala Ala Ala Gly Leu Thr Val Pro Ser Leu ThrMet Ala Pro Val Val Val Gly Ala Val Thr Leu Leu Ser Thr Val Phe Gly AlaGly Leu Val Leu Leu: (BGT2): Asp Asp Val Thr Pro Ile SEQ ID NO:3 Thr ValLys Gly Asn Ala Phe Phe Lys Gly Asp Glu Arg Phe Tyr Ile Arg Gly Val AspTyr Gln Pro Gly Gly Ser Ser Asp Leu Ala Asp Pro Ile Ala Asp Ala Asp GlyCys Lys Arg Asp Ile Ala Lys Phe Lys Glu Leu Gly Leu Asn Thr Ile Arg ValTyr Ser Val Asp Asn Ser Lys Asn His Asp Glu Cys Met Asn Thr Leu Ala AspAla Gly Ile Tyr Leu Val Leu Asp Val Asn Thr Pro Lys Tyr Ser Ile Asn ArgAla Lys Pro Lys Glu Ser Tyr Asn Asp Val Tyr Leu Gln Tyr Ile Phe Ala ThrVal Asp Ala Phe Ala Gly Tyr Lys Asn Thr Leu Ala Phe Phe Ser Gly Asn GluVal Ile Asn Asp Gly Pro Ser Ser Ser Ala Ala Pro Tyr Val Lys Ala Val ThrArg Asp Leu Arg Gln Tyr Ile Arg Ser Arg Lys Tyr Arg Glu Ile Pro Val GlyTyr Ser Ala Ala Asp Ile Asp Thr Asn Arg Leu Gln Met Ala Gln Tyr Met AsnCys Gly Ser Asp Asp Glu Arg Ser Asp Phe Phe Ala Phe Asn Asp Tyr Ser TrpCys Asp Pro Ser Ser Phe Lys Thr Ser Gly Trp Asp Gln Lys Val Lys Asn PheThr Gly Tyr Gly Leu Pro Leu Phe Leu Ser Glu Tyr Gly Cys Asn Thr Asn LysArg Gln Phe Gln Glu Val Ser Ser Leu Tyr Ser Thr Asp Met Thr Gly Val TyrSer Gly Gly Leu Val Tyr Glu Tyr Ser Gln Glu Ala Ser Asn Tyr Gly Leu ValGlu Ile Ser Gly Asn Asn Val Lys Glu Leu Pro Asp Phe Asp Ala Leu Lys ThrAla Phe Glu Lys Thr Ser Asn Pro Ser Gly Asp Gly Asn Tyr Asn Lys Thr GlyGly Ala Asn Pro Cys Pro Ala Lys Asp Ala Pro Asn Trp Asp Val Asp Asn AspAla Leu Pro Ala Ile Pro Glu Pro Ala Lys Lys Tyr Met Thr Glu Gly Ala GlyLys Gly Pro Gly Phe Ala Gly Pro Gly Ser Gln Asp Arg Gly Thr Gln Ser ThrAla Thr Ala Glu Pro Gly Ser Gly Ser Ala Thr Gly Ser Ser Ser Ser Gly ThrSer Thr Ser Ser Lys Gly Ala Ala Ala Gly Leu Thr Val Pro Ser Leu Thr MetAla Pro Val Val Val Gly Ala Val Thr Leu Leu Ser Thr Val Phe Gly Ala GlyLeu Val Leu Leu

This protein preferably has a molecular weight of about 44 kD, or ofabout 49 kD if it carries at least one residue of the N-glycosyl type.

The present invention also relates to proteins withβ-(1–3)-glucanosyltransferase activity characterized in that they haveat least 50%, preferably 60% and even more preferably 85% homology withproteins having the sequences, or a part of the sequences SEQ ID N^(o)10 or SEQ ID N^(o) 12 as follows:

Gly Phe Phe Ala Gly Asn Glu Val Ile Asn Glu Gln Ser Val Lys Asn SEQ IDNo 10 Val Pro Thr Tyr Val Arg Val Cys His Pro Ser Pro Gln Leu Thr IleAla Cys Pro Leu: (BGT4) Gly Phe Phe Ala Gly Asn Glu Val Val Asn Gln AlaAsn Gln Ser Ala SEQ ID No 12 Gly Ala Ala Phe Val Lys Ala Ala Ala Arg AspMet Lys Ala Tyr Ile Lys Thr Lys Gly Tyr Arg Gln Ser Leu Ala Ile Gly TyrAla Thr Thr Asp Asn Pro Glu Ile Arg Leu Pro Leu Ser Asp Tyr Leu Asn CysGly Asp Gln Ala Asp Ala Val Asp Phe Phe Gly Tyr Asn Ile Tyr Glu Trp CysGly Asp Gln Thr Phe Gln Thr Ser Gly Tyr Gln Asn Arg Thr Glu Glu Tyr LysAsp Tyr Ser Ile Pro Ile Phe Ile Ser Glu Tyr Gly Cys Asn

The present invention also relates to fragments of these proteins.

Said invention is not limited to the proteins having the sequences SEQID N^(o) 2, SEQ ID N^(o) 3, SEQ ID N^(o) 10 or SEQ ID N^(o) 12 butextends to any protein having sequences similar to those having thesequences SEQ ID N^(o) 2, SEQ ID N^(o) 3, SEQ ID N^(o) 10 or SEQ IDN^(o) 12 and in particular having certain amino acid substitutions inwhich an arnino acid is replaced by another amino acid havingessentially the same physico-chemical properties. Lehninger'sbiochemistry manual (Flarnmarion Medecine-Science, 1977, or one of itsmore recent editions) distinguishes four groups of amino acids, based ontheir physico-chemical behavior : those with a non-polar or hydrophobicside-chain, those with an uncharged polar side-chain, those with anegatively charged side-chain, and those with a positively chargedside-chain.

The present invention also relates to nucleotide sequences coding forproteins, or protein fragments such as those described above, and moreparticularly DNA sequences (cDNA or genomic DNA) or RNA sequences.

Such a DNA sequence may be that having at least 50%, preferably 60% andeven more preferably 85% homology with the genomic sequence SEQ ID N^(o)1, or a part of the sequence SEQ ID N^(o) 1 as follows:

ATG AAG GCC TCT GCT GTT ACT GCC GCT CTC GCC GTC GGT GCT TCC ACC GTT CTGGCA GCC CCC TCC ATC AAG GCT CGT GAC GAC GTT ACT CCC ATC ACT GTC AAG GGCAAT GCC TTC TTC AAG GGC GAT GAG CGT TTC TAT ATT CGC GGT GTC GAC TAC CAGCCC GGT GGC TCC TCC GAC CTG GCT GAT CCC ATC GCT GAT GCC GAT GGT TGC AAGCGT GAC ATT GCC AAG TTC AAG GAG CTG GGC CTG AAC ACT ATC CGT GTC TAC TCGGTC GAC AAC TCC AAG AAC CAC GAT GAG TGT ATG AAT ACA CTG GCT GAT GCT GGCATC TAT CTG GTG CTC GAT GTC AAC ACT CCC AAG TAC TCC ATC AAC CGC GCC AAGCCT AAG GAG TCG TAC AAC GAT GTC TAC CTC CAG TAT ATC TTC GCT ACC GTT GATGCT TTC GCC GGT TAC AAG AAC ACC CTC GCT TTC TTC TCC GGC AAC GAG GTT ATCAAC GAT GGC CCT TCC TCC TCT GCT GCT CCC TAC GTC AAG GCC GTC ACT CGT GATCTG CGT CAG TAC ATC CGT AGC CGC AAG TAC CGT GAG ATT CCT GTC GGC TAC TCGGCT GCC GAT ATC GAC ACC AAC CGT CTT CAG ATG GCC CAG TAT ATG AAC TGC GGTTCC GAC GAC GAG CGC AGT GAC TTC TTC GCT TTC AAC GAC TAC TCC TGG TGC GATCCC TCC TCT TTC AAA ACC TCG GGC TGG GAT CAG AAG GTC AAG AAC TTC ACT GGCTAC GGT CTT CCT CTC TTC CTG TCC GAA TAC GGC TGC AAC ACC AAC AAG CGT CAATTC CAA GAA GTC AGC TCT CTC TAC TCC ACG GAC ATG ACT GGT GTC TAC TCT GGTGGT CTC GTG TAC GAG TAC TCT CAG GAG GCC AGC AAC TAC GGT CTG GTG GAG ATTAGC GGC AAC AAT GTC AAG GAG CTC CCA GAC TTC GAC GCT CTG AAG ACC GCG TTCGAA AAG ACC TCC AAC CCC TCC GGC GAC GGC AAC TAC AAC AAG ACT GGT GGT GCCAAC CCT TGC CCC GCT AAG GAC GCT CCC AAC TGG GAC GTT GAC AAC GAT GCT CTTCCT GCC ATC CCC GAG CCC GCC AAG AAG TAC ATG ACT GAG GGT GCT GGC AAG GGCCCT GGT TTT GCC GGA CCT GGC AGC CAG GAC CGT GGT ACC CAG TCC ACT GCC ACTGCT GAG CCC GGA TCT GGC TCT GCC ACT GGA AGC AGC AGC AGC GGC ACC TCC ACCTCT TCC AAG GGC GCT GCA GCT GGC CTG ACT GTC CCT AGC CTG ACC ATG GCT CCCGTT GTC GTT GGT GCG GTT ACA CTC CTG TCC ACC GTC TTC GGC GCT GGC CTC GTCCTC TTG TGA

This sequence has been included in a 2.2 kb fragment, which has itselfbeen included in the X bal site of the pUC19 vector (Maniatis et at.,1989, Cold Spring Harbor Laboratories Press). The strain E. coli DH₅α acarrying this modified vector was deposited in the Collection Nationalede Culture de Micro-Organismes at the Institut Pasteur (CNCM) on the26th Jul. 1996 under the number I-1763.

Such a sequence may also be that having at least 50%, preferably 60% andeven more preferably 85% homology with the complementary DNA sequencecomprised in a 1.4 kb fragment, which has been included in the pCRIIvector (In Vitrogen). This, carried by the E. coli DH₅α strain, wasdeposited in the Collection Nationale de Culture de Micro-Organismes atthe Institut Pasteur (CNCM) on the 26th Jul. 1996 under the numberI-1762.

These two strains are objects of the present invention.

Nucleotide sequences according to the present invention may also bethose having at least 50%, preferably 60%, and even more preferably 85%homology with one of the DNA sequences SEQ ID N^(o) 9 or SEQ ID N^(o) 11as follows:

GGCTTCTTCG CCGGCAACGA GGTTATCAAC GAGCAGAGTG TCAAGAACGT TCCCACTTAC SEQ IDNo 9 GTCCGGGTAT GTCATCCATC CCCACAGCTT ACGATTGCCT GTCCACTGAC ACTCTCGTAGGCGACTCAGC GTGACATGAA GGACTACTAC GCAAAGAACC TTGACCGCAG CATTCCTGTTGGCTATTCTG CTGCCGATAT TCGTCCCATC CTCATGGCAC CCCTCAACTA CTTCATGTGCGCTGACGATG CTAATTCCCA ATCGGACTTC TTCGGCCTCA ACTCCTACTC GTGGTGCGGCAACTCGTCCT ACACCAAGAG TGGCTACGAT GTCCTCACCA AGGACTTTGC CGACGCCTCTATCCCCGTCT TCATCTCCGA ATTCGGCTGC AACA: GGTTTCTTCG CCGGCAACGA GGTTGTGAATCAGGCGAATC AGTCCGCCGG CGCTGCATTC SEQ ID No 11 GTCAAGGCCG CCGCGCGAGACATGAAGGCC TACATCAAGA CCAAGGGATA CCGGCAATCG CTGGCAATTG GATACGCGACCACTGACAAC CCGGAAATCC GACTCCCGCT GTCCGACTAC CTCAACTGCG GCGACCAGGCCGACGCGGTC GACTTCTTCG GCTACAACAT CTACGAATGG TGCGGTGACA AGACCTTCCAGACCTCGGGC TACCAGAACC GCACCGAGGA GTACAAGGAC TACTCCATCC CCATCTTCATCTCCGAATAC GGCTGCAAC

These two sequences have been independently included in the pCRIIvectors and introduced into the E. coli DH₅α strain. These strains weredeposited in the Collection Nationale de Culture de Micro-Organismes atthe Institut Pasteur (CNCM) on the 22nd Aug. 1997 under the numbersI-1914 and I-1913.

A further object of the present invention is a method for the detectionof proteins with a strong homology with the sequence of the proteinBGT2.

The present invention thus relates to a method for detecting anucleotide sequence having at least 60% identity with the sequence SEQID N^(o) 1 in a biological sample containing nucleotide sequences,comprising the following steps:

a) placing the biological sample in contact with the nucleotide primersP3 and P4 having the sequences SEQ ID N^(o) 7 and SEQ ID N^(o) 8,respectively, as follows:

GSYTTCTTCK CYGGCAACGA GGTT: SEQ ID No 7 GTTGCAGCCG WATTCGGASA YGAA: SEQID No 8

-   -   the nucleotide sequences contained in the sample having been if        necessary put into a form enabling their hybridization under        conditions enabling the hybridization of the primers with the        nucleotide sequences.

b) amplification of the nucleotide sequences

c) revealing the amplification products, and

d) detection of the mutations by appropriate methods.

The proteins according to the invention may be obtained by purificationof an autolysate of Aspergillus fumigatus. The protein may be purifiedby four steps of ion-exchange chromatography and one step of gelfiltration.

Said proteins may also be obtained by genetic engineering methods. Forexample, the sequence SEQ ID N^(o) 1, if possible without its C-terminalpart, may be cloned in an appropriate vector, and expressed in anexpression system, such as the Pichia pastoris system, marketed by InVitrogen.

In this system, the sequence of the gene coding for the protein iscloned in an expression vector, then lineanized. Protoplasts originatingfrom P. pastoris are transformed with the linearized vector.

The clones, in which a recombination is performed and which replaces theaoxl sequence by the sequence of the gene of the protein which it isdesired to produce, are selected for their capacity to grow in ahistidine-deficient medium. A person skilled in the art may refer to“Manual of methods for expression of recombinant proteins in Pichiapasioris”, published by In Vitrogen.

The protein thus expressed, if possible secreted in the culture medium,is recovered by processes known to a person skilled in the art.

Such a protein may be used, in particular, for screening molecules toidentify their antifungal activity.

Thus, another object of the present invention is a process for screeningmolecules to identify their antifungal activity comprising the followingsteps:

-   -   placing together the molecules to be screened and the protein or        the protein fragment as described above, or coded by a sequence        such as described above, and    -   determining the effect of the molecule on said protein.

The determination of the effect of the molecules on said protein may beaccomplished by measuring the activity of the β-(1–3)glucanosyltransferase (BGT2). Such activity may be determined by placingsaid protein in the presence of a substrate on which it has an effect,which may be composed of laninarioligosaccharides comprising at least 10glucosyl radicals linked by β-(1–3) bonds. When the protein is active,it cleaves a part of the molecule and binds the fragment obtained ontothe non-reducing terminal of an uncleaved substrate molecule.

The product resulting from the activity of the protein is in the form ofcoupling products of two laminarioligosaccharides. This product may bedetected by any process allowing the separation of oligosaccharides withdifferent degrees of polymerization, in particular by chromatographicmethods, such as high-pressure liquid chromatography (HPLC) orthin-layer chromatography (TLC). This latter method, although lessprecise than the first, is the easier to use.

For the use of these chromatographic methods, a person skilled in theart may consult the following manual : Carbohydrate analysis:a practicalapproach. Chaplin and Kennedy, 1986 IRC Press, Oxford.

This detection method enables determination as to whether the moleculesdetected have antifungal activity.

These molecules having antifungal activity show effects on theβ-(1–3)glucanosyltransferase activity of said proteins. These effectsmay be for example the inhibition of this activity.

The present invention also relates to molecules having an effect on saidproteins, which may be detected by the process as described above, aswell as the use of these molecules to prepare a drug, or for thetreatment of diseases related to fungi in vertebrates and plants.

One of the advantages of the use of these molecules lies in the lowfrequency of appearance of resistant strains of the fungi, in contrastto other known antifungal molecules.

The present invention is illustrated, without being limited, by thefollowing examples:

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates an analysis of the SDS-PAGE type of the purified 49kDa protein: line a, molecular weight standards; line b, purified 49 kDaprotein (1.5 μg); line c, purified 49 kDa protein (1.5 μg) aftertreatment with N-glycosidase F; line d, N-glycosidase alone. Themolecular weights of the protein bands and N-glycosidase F are shown.

FIG. 2 represents an HPAEC-type analysis of the products arising fromthe incubation of the 49 kDa enzyme with the reducedlamninarioligosaccharides. The purified 49 kDa protein was incubatedwith 8 mM of reduced laminarioligosaccharides of size G₁₁, G₁₂, G₁₃ orG₁₄ (reduced laminariofigosaccharides with 11, 12, 13 or 14 glucoseresidues respectively) and the HPAEC profiles arising from these samplesare shown at time zero and 15 min, with the sizes of the main products.

FIG. 3 represents an HPAEC-type analysis of the products arising fromthe incubation of the 49 kDa enzyme with 8 mM of rG₁₆. The BPAECprofiles arising from these samples are shown at time zero, 30 min and120 min, with the sizes of the main products.

FIG. 4 illustrates the action of the transferase and the products formedfrom the reduced laminarioligosaccharides.

FIG. 5 shows the effect of varying concentrations of the substrates. The49 kDa transferase was incubated with 3 μM of [³H]-rG₁₁ (1×10⁶ cpm) plusvarying quantities of unmarked rG₁₁. The % of transfer was determined bycomparing the proportion of marking formed in the form of rG₆ andrG_(16.) The inset shows the same data with the concentration of thesubstrates presented in the form of a linear scale.

FIG. 6 illustrates the effect of pH on the transfer rate. The 49 kDatransferase was incubated with 8 μM of [³H]-rG₁₁ (1×10⁶ cpm). Thereaction rates were determined by measuring the quantity of markingformed in the form of the product rG_(16.) The buffers used were: ●,sodium citrate/citric acid; ∘, imidazole/citric acid; x, sodiumacetate/acetic acid;▪, Tris/glycine; ▴, phosphate/NaOH; □, Tris/aceticacid; Δ, glycineHCl.

FIG. 7 shows the sequence originating from the gene BGT2 from A.fumigatus (A) compared with genes PHR1 and GASl isolated from C.albicans (C) and S. cerevisiae (S). The underlined amino acid sequencecorresponds to the presumed intron.

FIG. 8 shows the growth rate of mutant 49 compared to that of the wildstrain. Where a pH value is stated, the culture was performed atcontrolled pH (4 or 7).

FIG. 9 illustrates the growth of the wild strain and that of the mutantΔ49 and the variation of the pH of the culture medium during the growthof the two strains.

FIGS. 10A–10C are comparisons of the sequences of the protein withsequence SEQ ID NO: 2 and the five following proteins: Phr 1, Gas 2p,Gas 3p, Gas 4p, and Gas 5p.

FIGS. 11 and 12 are restriction maps of the sequences of the geneshomologous with the BGT2 gene, named BGT4 and BGT3 respectively.

EXAMPLES EXPERIMENTAL PROCEDURES

1. Preparation of the Cell Wall and Autolysis.

The strain CBS 144-89 of type A. fumigatus (available from theCollection Centralbureau voor Schimmelculture) was grown in a 15 Lfermenter in 2% glucose, 1% mycopeptone (Biokar Diagnostics) plus 0.1%silicone antifungal 426R (Rhodorsil) at 25° C. (agitation at 500 r.p.m.,aeration 0.5 vol.vol⁻¹. min⁻¹. for 42 h). A culture which had grown for3 days in a 2L fermenter under the same conditions was used as theinoculun (8%(v/v)). The mycelia were collected by filtration undervacuum and ruptured by passing through a Dyno type mixer in the presenceof glass beads (W. A. Bachofen AG, Basel, Switzerland) (0.5–0.75 mmdiameter). The progression of the disruption of the cells was monitoredmicroscopically. The suspension of the ruptured mycelia was centrifuged(8000 g, 15 min) and the residue containing the cell walls was washed 3times with water and once with 50 mM Na acetate, pH 5.6 containing 5 mMNa azide, then resuspended in the same buffer (250 g wet weight per L ofbuffer) and incubated (agitation 200 r.p.m.) at 37° C. After 72 h, thesuspension was centrifuged (10000 g, 15 min) and the supernatant wasplaced in a dialysis tube, concentrated 5 to 10 times with polyethyleneglycol 20000, dialyzed against 5 mM Na acetate, pH 5.6, recentrifuged(10000 g, 15 min) and filtered (0.45 μm filter). This preparation issubsequently referred to as the autolysate.

2. Enzyme Purification

The fractions collected during each step of the chromatography weretested for enzymatic activity using the non-radioactive transferase test(see below). The dialyzed and concentrated autolysate was applied to a4×18 cm DEAE-SEPHAROSE FAST-FLOW column (Pharmacia) equilibrated with 5mM Na acetate, pH 5.6, and the column was eluted with a linear gradientup to 1 M NaCI (2000 ml) at a flow rate of 240 ml.hr⁻¹. The fractionscontaining transferase activity were collected, dialyzed against abuffer containing 10 mM β-mercaptoethanol, 5 mM EDTA, 10 mM Na acetate,pH 4.0, applied to a MONO S column (HR 5/5 Pharmacia), and eluted with alinear gradient of NaCI (0 to 300 mM in 40 min) at a flow rate of 0.8ml.min⁻¹. The fraction containing transferase was collected, dialyzedagainst 10 mM Tris/HCI, pH 7.0, and applied to a DEAE-5PW column (8×75mm, TosoHaas), and eluted with a linear gradient up to NaCI (0 to 300 mMin 60 min) with a flow rate of 0.75 ml.min⁻¹. The fractions containingtransferase activity were collected, dialyzed against a buffercontaining 10 mM β-mercaptoethanol, 5 mM EDTA 10 mM EDTA, 10 mM Naacetate, pH 4.0, and applied to a CM-5PW column (8×75 mm, TosoHaas), andeluted with a linear gradient of NaCl (0 to 300 mM in 60 min) with aflow rate of 0.8 ml.min⁻¹. The fractions containing transferase activitywere collected and concentrated by a speed-vac and fractionated on aSUPERDEX HR75 column (Pharmacia) equilibrated with 10 mM Tri/HCl, pH 7.0containing 150 mM NaCl, at a flow rate of 0.75 ml.min⁻¹. The fractionscontaining purified transferase were collected, dialyzed against 5 mM Nacitrate, pH 5.0, concentrated by speed-vac and stored at −20° C. untilused.

3. Transferase Assays

The enzyme fractions were assayed for transferase activity by incubationin 50 mM Na citrate, pH 5.0 at 37° C. (10μl volume per assay) with alaminarioligosaccharide reduced with borohydride (8 mM final) of atleast size G₁₀. Samples (3 μl) were taken at different times, 50 mM NaOHcooled in ice (47 μl) was added to terminate the reaction, and themixture was frozen until analyzed by high-performance anion-exchangechromatography (HPAEC). Since the peak intensities detected by PulsedElectrochemical Detector (PED) varied from day to day, the transferaseactivity was quantified by use of reduced laminarioligosaccharidesmarked with ³H as substrates and measurement of the appearance of themarking in the products after separation by HPAEC chromatography, usingthe on-line Radiomatic 150TR scintillation rate analysis apparatus(Packard). Except where otherwise mentioned, the assays for the enzymecharacterization studies were performed as above with 0.25 μg ofpurified transferase.

4. Colorimetric determinations

The β-glucanase activity was measured in the protein fractions by asugar reduction test using the reagent hydroxybenzoic acid hydrazidewith laminarin reduced by borohydride instead of carboxymethyl pachymanas substrate (Ram et al., 1988, Life Sci Adv., 7, 379–383). Theexo-p-glucanase/β-glucosidase activities were measured by incubating theenzyme fractions with p-nitrophenyl-β-D-glucopyranoside (Hartland etal., 1991, Proc. R. Soc. London B, 246, 155–160). The quantity of theproteins was estimated using the Biorad protein test according to themanufacturer's instructions, with bovine serum albumin as standard.

5. High-performance Anion-exchange Chromatography

The samples from the transferase tests were analyzed on a DionexCARBOPAC PA1 analytical column (4×250 mm) (with a Pa1 reference column)on a Dionex HPAEC-type system with pulsed electrochemical detection(PED-2 cell), fitted with a combination of pH-Ag/AgCl referenceelectrodes and using a potential of 0.4 V for the first 0.5 s ofdetection. The oligosaccharides were eluted under the followingconditions: flow rate 1 ml/mm, buffer A: 50 mM NaOH; buffer B: 500 mMsodium acetate in 50 mM NaOH; gradient 0 to 2 mm, 98% A 2% B(isocratic), 2 to 15 min 75% A 25% B (linear), 15 to 45 min 60% A 40% B(linear).

The laminarioligosaccharide standards were obtained from Seikagaku(Japan).

6. Thin-layer Chromatography (TLC)

The laminarioligosaccharides were revealed by thin-layer chromatographyon silica gel 60 (Kieselgel, Merck) using n-butano/acetic acid/water(2/1/1.5) as eluant and sulfuric orcinol coloration.

The degree of polymerization (dp) of the oligosaccharides was alsodetermined by HPAE-type chromatography using a pulsed electrochemicaldetector and an anion-exchange column (CARBO6PAC PA1, 4.6×250 mm,Dionex).

7. Preparation of Reduced Substrates

The laminarioligosaccharides were obtained by partial acid hydrolysis(6.5 M TFA, 15 min, 1000° C., followed by 1 M TFA, 45 min, 1000° C.) ofcurdlan (Serva). The TFA was removed by rotary evaporation in thepresence of methanol. The oligosaccha rides were reduced overnight withNaBH₄ (1:0.5 (w/w)) in 0.1 M NaOH at room temperature). The reduced endsof the laminarioligosaccharides marked with ³H were similarly preparedby reduction with NaB³H₄ (Amersham, 20–40 Ci/mmol, 10 mCi per mg ofoligosaccharide) overnight followed by a subsequent reduction by NaBH₄as before. The excess of NaBH₄ was destroyed by addition of acetic acidup to pH 5–6, and the borate salts were removed by rotary evaporation inthe presence of methanol. The reduced oligosaccharides were desalted bygel filtration on a SEPHADEX G15 column (1.2×80 cm, 8 ml.h⁻¹,equilibrated in water) and collected after detection by theorcinol-sulfuric acid method (Ashwell, 1966, Methods Enzymol, 8, 85–95).The laminarioligosaccharides were separated by HPAEC on a CARBOPAC PA1preparative column (9×250 mm, Dionex) with a Na acetate gradient 15 to350 mM in 50 mM NaOH (45 min) at a flow rate of 4 ml.min⁻¹. Theoligosaccharide fractions collected were neutralized with acetic acid,desalted by gel filtration on a SEPHADEX G15 column as described above,then lyophilized. The laminarin (Sigma) was reduced in the same way, butdesalted by dialysis against 0.5% acetic acid, followed by dialysisagainst water, then lyophilized. The gentiooligosaccharides wereprepared as above (without reduction) from pulsatin (Calbiochem) whichhad been finely divided with a pestle and mortar. The maltoheptaose andcellopentaose were from Boehringer Mannheim and Sigma, respectively. Thechitohexaose was a gift from Dr. A Domard (Université Claude Bernard,Villeurbanne, France). G₁₀ reduce with borohydride containing a β-(1–6)intrachain bond at the sixth link from the reduced end (rG₁₀*) wasobtained by incubating the reduced laminarihexose (rG₆) with an enzymehomologous with the BGT1 enzyme from Candida purified from A. fumigatus.The product from the transferase rG₁₀* was separated and purified as forthe laminarioligosaccharides.

7.Electrophoresis on SDS-polvacrylamide Gel

The protein samples were analyzed by SDS-PAGE (Laemili, 1970, Nature,227, 680–685) using 10% separation gels and 4% stacked gels. The proteinbands were revealed by coloration with Coomassie blue. TheN-glycosylation of the glycoproteins was performed using the recombinantN-glucosidase F (Oxford GlycoSystems) according to the manufacturer'sinstructions.

8. ¹H NMR Spectroscopy

Two samples were analyzed the reduced laminarioligosaccharide G₁₀ usedas standard and a reduced oligosaccharide G₁₆ obtained after incubationof rG₁₀ with the transferase and purified by HPAEC. The deuterium in thesamples dried by lyophilization was replaced by dissolution in D₂O(99.95%, Solvents Documentation Synthese, France). The spectra wererecorded at 300 K and 318 K on Variant Unity 500 spectrometer operatingat a proton frequency of 500 MHz. The OH resonance of the residual waterwas removed by selective radiation during the relaxation time. Sodium3-trimethylsilylpropionic acid was used as external standard.

EXAMPLE 1 Purification of the 49 kDa Protein

A high-performance anion-exchange chromatography (HPAEC) test usinglaminarioligosaccharides reduced with borohydride as substrates wasdeveloped to study the activities of the β-glucanosyl transferaseassociated with the cell wall of A. fumigatus. A new β-(1–3)-glucosyltransferase activity was detected in the semi-purified fractions fromthe autolysate of the cell wall of A. fumigatus, which remainedassociated with a 49 kDa protein throughout its purification.

The protein was purified to apparent homogeneity with four steps ofion-exchange chromatography and one gel filtration step.

The activity of the transferase was clearly detectable after only thesecond chromatography step (MONO S). The analysis by SDS-PAGE of thepurified fraction showed a main band at 49 kDa (FIG. 1, well b). Inorder to determine if it contained an N-linked carbohydrate, the proteinwas digested with N-glucosidase F. The digested protein passed onSDS-PAGE as 44 kDa protein, (FIG. 1, well c) showing that it containedabout 5 kDa of N-linked carbohydrate.

EXAMPLE 2 Enzymatic Activity of the 49 kDa Protein

HPAEC analysis of the products resulting from the incubation of the 49kDa protein with a larninarioligosaccharide reduced with borohydride(rG_(n)) of size G₁₀ or larger led to the characterization of a newactivity of glucanosyl transferase type.

The principal initial products arising from the incubation with rG₁₁were rG₆ and rG₁₆, rG₁₂ gave rG₆+rG₇ and rG₁₇+rG₁₈, rG₁₃ gave rG₁₈ torG₂₀, and rG₁₄ gave rG₆ to rG₉ and rG₁₉ to rG₂₂ (FIG. 2). Significantly,no products of the larninarioligosaccharide type, reduced or not, weredetected, confirming the absence of any endo-β-(1–3)-glucanase activity.The presence of such activity would have caused the formation of amixture of hydrolysis products, reduced or not, the latter havingdifferent retention times. In addition, no glucose was detected, andtogether with the absence of hydrolysis ofp-nitrophenyl-β-glucopyranoside and the formation in the network ofreducing sugar arising from the laminarin reduced with borohydride inthe corresponding calorimetric tests, this confirmed the absence ofexo-β-(1–3)-glucanase and β-glucosidase activity.

The profile of the products obtained (FIG. 2) is in agreement with anendogenous type of glucanosyl transferase activity in which the glucanchain is cleaved by an endolytic cleavage, freeing the portion of thereduced end, and the remainder is transferred to another glucan chain,to form a larger transferase product. Thus, in the simplest reactionwith rG₁₁, the enzyme cleaves the substrate, liberating rG₆ from thereduced end of the substrate molecule, and the remaining G₅ is thentransferred to another rG₁₁ molecule acting as receptor, to form atransferase-type product rG₁₆:

-   -   E+rG₁₁→E. G₅+rG₆    -   E. G₅+rG₁₁→E+rG₁₆        where E represents the enzyme. The transferase cleaves rG₁₂ in        two different places, leading to two different transferase-type        products:    -   E+rG₁₁→E. G₅+rG₆        -   →E. G₅+rG₇    -   E. G₆+rG₁₂→E+rG₁₈    -   E. G₅+rG₁₂→E+rG₁₇

Similarly with rG₁₃ and rG_(14,) the transferase cleaves in three orfour different places, respectively, each time transferring the part ofthe non-reduced end to another acceptor molecule rG₁₃ or rG_(14.)

Additional analyses of incubations of the 49 kDa transferase withreduced or smaller laminarioligosaccharides showed that the reactionwith rG₁₀ gave rG₁₀+rG₆ and rG₁₄+rG_(15,) as initial major products,while the reaction with rGg was extremely slow, forming small peaks ofrG₅ to rG₈ and rG₁₀to rG_(13.) No products were detected afterincubation with laminarioligosaccharides of size G₈ or smaller.

In order to determine the relative reaction rate of the enzymes withlarninarioligosaccharides of variable sizes, the 49 kDa enzyme (0.25 μg)was incubated with 8 mM of rG₁₀ to rG₁₅ marked with ³H and the rate offormation of the marked products was measured. The rate with rG₁₀ (328nmol.min⁻¹) was approximately equal to 50% of that with the largersubstrates and there was no significant difference between the reactionrates for rG₁₁ to rG₁₅ (648±46 nmol.min⁻¹.mg proteins⁻¹).

Analysis of longer incubations of the purified enzymes with reducedlaminarioligosaccharides of size at least G₁₀ showed that the productsfrom the initial transferase could be re-used either as donors or asacceptors, leading to the formation of products of increasing size,until they are eliminated from the solution because of theirinsolubility in the aqueous buffer. An incubation of 30 min with rG₁₆(containing some contaminating rG₁₅) led to the formation of reducedinitial major products of sizes G₆ to G₁₁ and G₂₁ to G₂₆ (FIG. 3), butafter 120 min, larger transferase products appeared with sizes of atleast G₄₀ (FIG. 3). The products with sizes G₂₉ and larger precipitatedat the bottom of the incubation tube since they were absent when thereaction mixture was briefly centrifuged and the supernatant analyzed.Incubation of the purified enzyme with reduced larninarin led to theproduction of smaller and larger products, showing that solubleoligosaccharides of size at least G₃₀ and larger can act as donors andacceptors in the reaction.

In order to determine the smallest laminarioligosaccharide which couldact as acceptor, the purified transferase was incubated with 4 mM ofrG₁₁ as donor and 16 mM of rG₈ or smaller as acceptor. Analyses of theincubations of rG₁₁ rG₄ or smaller showed the formation of rG₆ and rG₁₆as the only initial major products, showing that only rG₁₁ had been usedas acceptor. However, incubations containing rG₁₁ and rG₅ to rG₈ showedadditional transferase products consistent with the use of the latteroligosaccharides as acceptors. For example, the reaction of rG₁₁ and rG₇led to the initial formation of rG_(6,) rG₁₂ and rG₁₆ consistent with:

-   -   E+rG₁₁→E.G₅+rG₆    -   E.G₅+rG₁₁→E+rG₁₆    -   E.G₅+rG₇→E+rG₁₂

The relative rate of the reaction with the acceptors was determined byusing 2 mM of rG₁₁ as donor and 32 mM of reduced acceptor marked with ³Hand measuring the formation of marked transferase product (FIG. 4).Under these conditions, the reaction with rG₁₁ used as acceptor wasnegligible. The reaction rate increased with an increase in chainlength, showing that the 49 kDa enzyne prefers the largerlaminarioligosaccharide acceptors.

The transferase showed no activity towards the gentiooligosaccharides(size G₃₋₈), chitohexaose, cellopentaose or maltoheptaose, either in thepresence or absence of rG₁₁ suggesting that the enzyme exclusively usesa β-(1–3) glucan as donor. This was demonstrated by using a reducedbranched G₁₀ (rG₁₀*) similar to the laminaridecaose, except that thesixth link from the reduced end is a β-(1–6)-type link. Incubation ofthe 49 kDa enzyme with 8 mM of rG₁₀* gave no products, showing that itwas not a donor. However, a similar incubation in the presence of 2 mMof rG₁₁ led to the formation of rG₆ and an elution peak in the positionof rG₁₅ as initial major products, showing that rG,₁₀* can act as anacceptor.

EXAMPLE 3 ¹H NMR Analysis of the Reduced G₁₆ Transferase Product

In order to determine if the 49 kDa transferase had produced a new typeof bond during the transfer, the product rG₁₆ of the transferase waspurified from the incubation medium of the transferase with rG₁₁.Approximately 300 μg of the product were analyzed by ¹H NMR The lDspectrum of the product rG₁₆ of the transferase showed three chemicalshifts in the anomeric region:

-   -   δ=4.68 ppm corresponding to the glucose residue linked to the        glucitol group;    -   δ=4.75 ppm corresponding to the glucose residue of the        non-reduced end;    -   δ=4.80 ppm corresponding to the intrachain residues of glucose,        linked β-(1–3).

The relative intensities of the anomeric signals showed 1, 1 and 13protons respectively. Since the glucitol gives no signals in theanomeric region, this confirms the length of the oligosaccharide (16residues), The coupling constants measured for these signals were inagreement with β-linked glucose residues (³J_(1,2)=7.9 Hz). The presenceof a single unit of the glucose type at the non-reduced end indicatesthat the 49 kDa protein had been transferred to the non-reduced end ofthe β-(1–3) glucan acceptor.

The 1D spectrum of the product rG₁₆ was identical, except for therelative intensity of the 4.80 ppm signal compared to that of the rG₁₀larninarioligosaccharide standard. In addition, no chemical shiftcharacteristic of a glucose residue linked (1–2), (1–4) or (1–6) wasvisible, confirming that the rGI6 product was a larninari-hexadecaose.The conjoint elution of the rG₁₆ product with the rG₁₆ reference onHPAEC and the insolubility of the larger product are in agreement withthe production of a β-(1–3)-type bond during the transfer.

EXAMPLE 4 Effect of Substrate Concentration on the Reaction Products

In order to determine whether the concentration decreasing of acceptorsstimulated the hydrolysis reactions, the 49 kDa transferase wasincubated with 3 μl of [³H]-rG₁₁ and decreasing quantities of unmarkedrG₁₁. A shift from the transfer (i) to the hydrolysis (ii) was observed(FIG. 5, inset):

-   -   E+[³H]-rG₁₁→E.G₅+[³H]-rG₆    -   (i)→E.G₅+[³H]-rG₁₁→E+[³H]-rG₁₆    -   (ii)→E.G₅+H₂O→E+G₅        The percentage of transfer was determined by measuring the        formation of marked rG₁₆ (transfer only) compared with that of        marked rG₆ (transfer plus hydrolysis) in the reaction. At an        rG₁₁ concentration of 3 mM, only transfer was detected. As the        substrate concentration reduced to 18 μM, the percentage of        transfer leveled out at about 35% and did not decrease        significantly with a low substrate concentration (3 μM)(FIG. 5).        Reduction of the buffer concentration to 10 mM did not change        the transfer percentage for any substrate concentration. It        seems that below the given conditions, the 49 kDa transferase        was unable to catalyze more than about 65% of hydrolysis by        simply reducing the substrate concentration to very low levels.

EXAMPLE 5 Optimum pH1 and Stability

The 49 kDa enzyme was tested at different pH values, the storagestability was verified and the activity of the N-glycosylated enzyme wastested. The enzyme was active over a wide range of acid pH, showing anactivity of more than 50% of its maximum between pH 2.5 and 6.0. Theenzyme showed a pH optimum of about 5.0 in citrate buffer (FIG. 6). Theenzyme was very stable and could be stored at 4° C. in 10 mM citratebuffer, pH 5.0 for several weeks, or dried by a speed-vac and thenre-suspended in a buffer, or stored at −20° C. without significant lossof activity. The 44 kDa de-N-glycosylated enzyme prepared undernon-denaturing conditions was as active as the native glycosylatedenzyme when incubated with 8 mM of rG₁₁.

EXAMPLE 6 Kinetic Analysis

The 49 kDa enzyme catalyzed its transferase-type reaction by abi-reaction type mechanism (two steps) with an initial hydrolysis of thesubstrate to liberate the portion of the reduced end, and a subsequenttransfer of the remainder of the non-reduced end to a substrate moleculeplaying the role of an acceptor molecule.

By using rG₁₁ as substrate, it was impossible to calculate an apparentKm corresponding to the two steps of the reaction. In order to determinea Km for the donor site, we used an acceptor which was not a donor. Weused [³H]-rG₇ (1×10⁶ cpm) as acceptor at a high concentration (64 mM)with different concentrations of rG₁₁ kept below 8 mM. Under theseconditions, the reaction took place with rG₁₁ as donor and rG₇ was usedrather than rG₁₁ as acceptor, as determined by the absence of formationof the transferase product rG₁₆. The initial reaction rate wasdetermined by measuring the appearance of marked rG_(12.)

An apparent Km of 5.3 mM was obtained from reciprocal double spots(value of r²=0.997).

EXAMPLE 7 Cloning, Sequencing and Interruption of the Gene Coding forBGT2 in A. fumigatas.

Two amino acid sequences were obtained from the purified BGT2 protein.The NH₂ terminal sequence was DVTPITVKGNAFFKGDERFY (SEQ ID NO: 20) andan internal sequence was DAPNWDVDNDALP (SEQ ID NO: 21). Anoligonucleotide of 38 units on the N-terminal part having the followingsequence SEQ ID N^(o) 4: (AAG GG(T/C) AA(C/T) GC(T/C) TTC TT(C/T) AAGGG(T/C) GA(T/C) GAG CG(T/C) TTC TA) was used to screen a gene bankcreated in the phage EMBL3 after partial digestion by Sau3A of the DNAof A. fumigatus as described by Monod (1994, 33–40, Mol. Biology ofpathogenic fungi, B. Maresca and G. S. Kobayashi).

The transfer was performed on membranes of the ZETAPROBE type. Themembranes were pre-hybridized and hybridized at 500° C. in a solutioncontaining SSC 5×, Na₂HPO₄25 mM, pH7, SDS 7%, Denhard 10× and 1% salmonsperm. The membranes were washed twice at 42° C. in a solutioncontaining SSC 3×, Denhard 10×, SDS 5%, Na₂HPO₄25 mM, and twice in anSDS solution with 1% SSC 1×.

The cloning and sequencing of the gene coding for the BGT2 proteinshowed significant homologies with the genes PHR1 and GAS1 previouslyidentified in C. albicans and S. cerevisiae respectively (Saporito Irwinand Coil. (1995) Mol. Cell Biol., 15, 601–613; Nuoffer and Coil., J.Biol. Chem., (1991), 226, 19, 12242–12248) (FIG. 7). The GAS 1 gene fromS. cerevisiae was also responsible for a glucanosyl transferaseactivity. In this fungus species, the minimum size of optimal substratewas G_(10,) rather than G₁₁ for A. fumigatus.

The disruption of the BGT2 gene was carried out by using the vectorpAN7–1 (Punt and Coil. (1987) Gene 56, 117–124) supplied by P. Punt(TNO, Rijinsik). This vector was modified as pN4 (Paris and Coil. (1993)FEMS Microbiol. Lett. 111, 31–36) by the replacement of a restrictionsite HindIII by a Smal site. About 50% of the open reading frame of BGT2was replaced by pN4 at an EcoRV restriction site. A completetransformation was performed as previously described (Paris, (1994)Isolation of protease negative mutants of Aspergillus fumigatus byinsertion of a disrupted gene, p. 49–55. Mol. Biology of pathogenicfungi, B. Maresca and G. S. Kobayashi) by using protoplasts produced byNovozyme and the linearized plasmid in the presence of PEG.

The Δ49 mutant from A. fumigalus obtained was deposited on 30th Jul.1996 at the CNCM under the deposit number I-1764.

It showed no phenotype distinct from the wild strain, except for a totalinhibition of the growth in the fermenter after 24 hours growth.

EXAMPLE 8 Cloning and Sequencing of the cDNA of BGT2.

The cDNA of BGT2 was obtained by amplification with two primers of cDNAtype 5′ GAATTCGACGACGTTACTCCCATCACT 3′ (SEQ ID NO: 5) of P1 and 5′TCTAGAGGGTATGAGAAGAACAAATCA 3′ (SEQ ID NO: 6) of P2 obtained from 10 ngof cDNA, 1 U of taq polymerase, 200 mM of each primer. 30 Amplificationcycles were performed, comprising 1 minute at 95° C., one minute at 55°C. and one minute at 72° C.

The amplified preparation was then cloned in a vector using a TA Cloningkit (In Vitrogen).

EXAMPLE 9 Expression of the β-(1–3)-glucanosyltransferase

Experiments using Triton X114 divisions with or without treatment withGPI-phospholipase C showed that the protein BGT2 from A. fumigatus wasattached to the plasma membrane by a GPI residue.

The attachment of the protein to the membrane was not necessary forretention of enzymatic activity. We showed that in A. fumigatus the sameactivity was present when the protein was either free in the culturemedium (in the absence of the GPI bond) or attached to the plasmamembrane.

These results suggested that the expression of the glucanosyltransferasecould be performed in vectors by a secreted expression. The Pichiapastoris expression system from In Vitrogen was selected. This systemhad been previously used for another 88 kDA protein from A. fumigatusand it was confirmed that Pichia preserved the glycosylation site of thenative protein very well.

The vector used was pPICZα (In Vitrogen) for the secretion with a mycepitope and six histidine residues in tandem for easy purification. TheC-terminal sequence responsible for the attachment by GPI was removedbefore the sub-cloning in pPICZα so as to obtain an enzymaticaldy activetruncated secreted protein.

This recombinant protein was used for the detection of antifungal drugs.The inhibition of the enzymatic activity may be measured by an HPLC-typedetection in the absence of cleavage and an additional elongation of anyβ-(1–3) laninarioligosaccharides with dp >10 in the presence of p49 fromA. fumigatus.

The absence of motility of the laminarioligosaccharide measured bythin-layer chromatography by conventional techniques or directly aftermarking of the reduced end with a chromogenic or fluorogenic radicalcould be a fundamental technique for performing detection automatically.

Since the product from the β-(1–3) glucanosyltransferase becomesinsoluble in an aqueous medium, because of the elongation of theβ-glucan chain, the absence of precipitation of any product afterprolonged incubation using a radioactively marked substrate could alsobe used for drug detection.

EXAMPLE 10 Identification of Genes with Homologies with BGT2

Degenerate oligonucleotide primers corresponding to the regions retainedin the sequence in FIG. 10 were synthesized by GENSET. They had thefollowing sequences:

(P3) 5′ GSYTTCTTCKCYGGCAACGAGGTT 3′: SEQ ID No 7 (P4′)5′ GTTGCAGCCGWATTCGGASAYGAA 3′: SEQ ID No 8in which Y is C or T

-   -   K is T or G    -   S is C or G    -   W is A or T.

PCR reactions were carried out in a volume of 100 μl containing 1.5 mMMgCl₂, 50mM KCI, 10 mM TrisCl (pH 8), 250 μM of dATP, dGTP, dCTP anddTTP (Pharmacia), 1 μM of each primer, 2.5 units of AMPLITAQ DNApolymerase (Pharmacia) and 50 ng of genomic DNA. The amplification wasperformed in an OmniGene apparatus initially for 5 min at 93° C., thenfor 30 cycles of 1 min at 93° C., then for 1 min at 50° C. and 1 min at72° C. The products from the PCR reaction were analyzed byelectrophoresis on 1% agarose gel, then revealed by ultraviolet aflercoloration with ethidium bromide.

The fragments resulting from the PCR reaction were ligatured in pCR2.1(TA cloning kit, In Vitrogen). The recombinant plasmid inserts weresequenced by the dideoxy chain termination method (Sanger et al., 1977)using SEQUENASE, Version 2 (US Biochemicais) according to themanufacturer's instructions.

Two nucleotide sequences were thus amplified : sequences SEQ ID N^(o) 9and SEQ ID N^(o) 11. The amino acid sequences deduced from thenucleotide sequences were sequences SEQ ID N^(o) 10 and SEQ ID N^(o) 12,corresponding to the genes named BGT4 and BGT3.

These sequences are in particular described in the list below. They haveidentity percentages with BGT2 of 41% and 37% respectively.

Restriction maps of the two sequences SEQ ID N^(o) 10 and SEQ ID N^(o)12 are represented respectively in FIGS. 12 and 11.

The sequences SEQ ID N^(o) 9 and SEQ ID N^(o) 11 were inserted into theplasmid PCRII which was introduced into E. coli. These bacteria weredeposited on the 22nd Aug. 1997 at the CNCM under numbers I-1914 andI-1913 respectively.

TABLE Reaction of the transferase with an initial rate of formation ofthe transfer product Transfer rate Transferase reaction (nmol · min⁻¹ ·mg protein⁻¹) rG₁₁ + [³H]-rG₅ → RG₆ + [³H]-rG₁₀ 203 rG₁₁ + [³H]-rG₆ →RG₆ + [³H]-rG₁₁ 387 rG₁₁ + [³H]-rG₇ → RG₆ + [³H]-rG₁₂ 484 rG₁₁ +[³H]-rG₈ → RG₆ + [³H]-rG₁₃ 586

1. A process for detecting antifungal activity of a molecule comprising:combining a molecule to be tested with a protein havingβ-(1–3)-glycanosyltransferase type activity, wherein the proteincomprises SEQ ID NO:2, and determining an effect of the molecule on theprotein.
 2. The process according to claim 1, wherein the proteinexhibits a molecular weight of 44 kD.
 3. The process according to claim1, wherein the protein comprises N-linked carbohydrates.
 4. The processaccording to claim 1, wherein the protein exhibits a molecular weight of49 kD.
 5. The process according to claim 1, wherein the protein isattached to a plasma membrane by a glycosylphosphatidylinositol radical.6. The process according to claim 1, wherein the effect of the moleculeon the protein is determined by measuring theβ-(1–3)-glycanosyltransferase type activity.
 7. The process according toclaim 1, wherein the μ-(1–3)-glycanosyltransferase type activity isdetermined by placing the protein in the presence of a substrate onwhich it has activity.
 8. The process according to claim 7, wherein thesubstrate comprises at least one laminarioligosaccharide comprising atleast ten glucosyl radicals.
 9. The process according to claim 7,wherein the activity of the protein on the substrate is detected usingchromatography.
 10. The process according to claim 9, wherein thechromatography is chosen from high pressure liquid chromatography andthin layer chromatography.
 11. The process according to claim 9, whereinthe chromatography separates oligosaccharides with different degrees ofpolymerization.